DESCRIPTION: This proposal focuses on the mitogen-activated protein (MAP) kinase cascade leading to activation of ERK1 and ERK2. This pathway controls cell growth, differentiation, and development through growth factor-regulated intracellular phosphorylation. Hyperactivation of this pathway leads to cell transformation, whereas pathway lesions lead to arrested growth and development in various organisms. Thus, it is a critically important target for therapeutic intervention. Regulators of ERKs are the MAP kinase kinases, MKK1 and MKK2, which activate ERKs by direct phosphorylation. MKK1 and MKK2 are in turn activated by at least three upstream kinases, Raf-1, Mos, and MLK-3, through phosphorylation at two primary sites. Secondary phosphorylation sites are also observed. Dr. Ahn's preliminary data show that these secondary phosphorylation sites modulate MKK activity in mammalian cells, thus influencing downstream signaling and cell transformation. The overall aim of this proposal is to examine biochemical mechanisms that regulate MKK1 and MKK2 and subsequent signaling through the MAP kinase cascade. Although a general model exists for phosphorylation and activation of MKK by upstream kinases, the mechanisms that tune the pathway remain obscure. For example, it is becoming apparent that the extent and kinetics of MKK/ERK activation as well as their intracellular localization play a vital role in determining biological responses to this pathway. Yet, specific mechanisms to account for the localization and signaling profiles are lacking. Underlying these cellular aspects of pathway regulation are the enzymological mechanisms controlling MKK or ERK activation. Again, an understanding of the conformational changes that switch protein kinases from inactive to active states is lacking. In this grant Dr. Ahn proposes several approaches to obtain a more complete mechanistic understanding of MKK1 and 2 regulation. The specific aims are to (i) investigate the means by which MKK1 and MKK2 phosphorylation at secondary sites modulate activity in vivo, focusing on how phosphorylation controls interactions with, and regulation by, other signaling molecules; (ii) examine structural features of MKK1 and 2 involved in controlling nuclear import, and the importance of nuclear translocation on signaling; and (iii) study conformational changes in MKK1 that occur upon activation, comparing effects of phosphorylation vs. mutagenesis, to define conformational requirements for kinase activation.